Recent advances in bioanalytical sciences and bioengineering have led to the development of DNA chips, miniaturized biosensors, and microfluidic devices. In addition, applications benefiting from fluorescent labeling include medical (and non-medical) fluorescence microscopy, histology, flow cytometry, fundamental cellular and molecular biology protocols, fluorescence in situ hybridization, DNA sequencing, immunoassays, binding assays and separation. These enabling technologies have substantially impacted many areas in biomedical research, such as gene expression profiling, drag discovery, and clinical diagnostics.
Fluorescently-labeled molecules have been used extensively for a wide range of applications. Typically organic dyes are bonded to a probe, which in turn selectively binds to a target molecule. Then the target molecule is identified by exciting the dye molecule, causing it to fluoresce. There are many disadvantages to using an organic dye for these fluorescent-labeling systems. The emission of visible light from an excited dye molecule usually is characterized by the presence of a broad emission spectrum (about 100 nm) and broad tails of emission at red wavelengths (about another 100 nm). As a result, there is a severe limitation on the number of different color organic dye molecules which can be utilized simultaneously or sequentially in an analysis since it is difficult to either simultaneously or even non-simultaneously detect or discriminate between the presence of a number of different detectable substances due to the broad spectrum emissions and emission tails of the labeling molecules.
Another problem is that organic dyes often have a narrow absorption spectrum (about 30-50 nm), thus requiring either multiple wavelength probes, or else a broad spectrum excitation source, which is sequentially used with different filters for sequential excitation of a series of probes respectively excited at different wavelengths. An additional problem associated with organic dyes is their lack of photostability. Often organic dyes bleach or cease to fluoresce under repeated excitation.
Therefore, there is a need in the industry for more accurate, sensitive, and broader method of detection.